CYCLIC TRIAZO SODIUM CHANNEL BLOCKERS

Description

[0001] The present invention relates to triazine compounds having sodium channel blocking properties, and to use of the compounds for preparation of medicaments for treatment of associated disorders.

[0002] US Patent No. 4,649,139 discloses compounds of the formula (A):

in which R¹ is C_1-10 alkyl, C_2-10 alkenyl, C_2-10 alkynyl or C_3-10 cycloalkyl, any of which is optionally substituted, and R² to R⁶ are independently selected from hydrogen, halogen, C_1-6 alkyl, alkenyl, alkynyl or alkoxy (all optionally substituted by one or more of halogen, hydroxy and aryl), amino, mono- or di-substituted amino, alkenyloxy, acyl, acyloxy, cyano, nitro, aryl and alkylthio groups or any adjacent two of R² to R⁶ are linked to form a (-CH=CH-CH=CH-) group. It is disclosed that these compounds are active in the treatment of cardiac disorders, and are particularly useful in the treatment of arrhythmias.

[0003] Our previous patent application WO2008/007149 discloses uses of a compound of formula (B):

in which R¹ is hydrogen (and =NH is NH₂), or is carboxamido, C_1-10 alkyl, C_2-10 alkenyl, C_1-3 alkyl-aryl, C_1-3 alkyl-heterocyclyl, or C_3-10 cycloalkyl, any of which is optionally substituted by hydroxy, halogen, carboxamido, halo C_1-6 alkyl, C_1-6 alkyl or C_1-6 alkoxy; and R² to R⁶ are independently selected from hydrogen, halogen, C_1-6 alkyl, alkenyl, alkynyl or alkoxy (all optionally substituted by one or more of halogen, hydroxy and aryl), amino, mono- or di-substituted amino, alkenyloxy, acyl, acyloxy, cyano, nitro, aryl and alkylthio groups;

(a) as voltage-dependent sodium channel blockers for the treatment of disorders in mammals, and particularly epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias, especially in humans; and

(b) as antifolates for the treatment of disorders in mammals, and particularly for treatment of mammalian cancers and as antimalarials against plasmodium vivax and plasmodium falciparum malaria, especially in humans.

[0004] According to the present invention there is provided the compound of formula (I):

in which Z is a single bond and wherein A is 2,3-dichlorophenyl and R¹ is -CH₂CCl₃ ; or a salt thereof.

[0005] The present invention also provides salts of the above compound. Preferred salts are pharmaceutically acceptable acid addition salts. Suitable pharmaceutically acceptable acid addition salts include those formed with both organic and inorganic acids, for example from hydrochloric, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, malonic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, p-toluenesulphonic, benzenesulphonic, glutamic, naphthoic, and isethionic acids. Ethanesulphonate, malate, mandalate, benzoate, and salicylate salts are also suitable.

[0006] The present invention also provides solvates of the compound of formula (I) or salts thereof. The compound or its salt may be obtained as a solvate of the reaction solvent or crystallisation solvent or a component thereof in preparation of the compound. Suitable pharmaceutically acceptable solvates include hydrates.

[0007] The present invention encompasses tautomeric forms and mixtures thereof, and polymorphic crystalline forms of the compound of formula (I).

[0008] The compound of formula (I) and certain analogues may be prepared by the procedures disclosed in the above-mentioned US Patent No. 4,649,139, to which further reference should be made. The compound of formula (I) and certain analogues may also be prepared by methods disclosed in EP 0 021 121 A, to which further reference should be made.

[0009] The preparation of the compound or formula (I) and certain analogues is illustrated later in this specification. Related compounds may be prepared by obvious or routine variations of the disclosed processes, using appropriate starting materials to introduce the desired substituents and moieties of analogues to the compound of formula (I).

[0010] Salts of the compound of formula (I) may be obtained by the presence of a residual acid in the preparative process. Alternatively salts may be prepared by mixing the compound of formula (I) as the free base with a pharmaceutically acceptable acid in a suitable solvent, and removing the solvent to recover the salt, or crystallising the salt from the solvent.

[0011] In a further aspect, the present invention provides pharmaceutical compositions comprising the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable carrier. The compounds are suitable for the treatment of disorders such as epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood,anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for treatment of mammalian cancers; and for treatment of malaria.

[0012] The compound of formula (I) is present in the compositions of the present invention in an effective unit dosage form, that is to say in an amount sufficient to be effective against the disorders in vivo.

[0013] The pharmaceutically acceptable carriers present in the compositions of the present invention may be materials conventionally used for the purpose of administering the medicament. These may be liquid or solid materials, which are otherwise inert or medically acceptable and are compatible with the active ingredients.

[0014] These pharmaceutical compositions may be given orally or parenterally, for example as a suppository, ointment, cream, powder or trans-dermal patch. However, oral administration and intravenous injection of the compositions are preferred.

[0015] For oral administration, fine powders or granules will contain diluting, dispersing and/or surface active agents, and may be presented in draught, in water or in a syrup, in capsules or sachets in the dry state or in non-aqueous suspension wherein suspending agents may be included, or in a suspension in water or syrup. Where desirable or necessary, flavouring, preserving, suspending, or thickening agents can be included. Dry powders or granules may be compressed to form a tablet or contained in a capsule.

[0016] For injection, the compounds may be presented in sterile aqueous injection solutions which may contain anti-oxidants or buffers.

[0017] The free base or a salt or solvate thereof may also be administered in its pure form unassociated with other additives in which case a capsule or sachet is the preferred carrier.

[0018] Alternatively the active compound is presented in a pure form at an effective unit dosage, for instance compressed as a tablet or the like.

[0019] Other compounds which may be included are, for example, medically inert ingredients, e.g., solid and liquid diluents such as lactose, starch, or calcium phosphate for tablet or capsules; olive oil or ethyl oleate for soft capsules; and water or vegetable oil for suspensions or emulsions; lubricating agents such as talc or magnesium stearate; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate; and other therapeutically acceptable accessory ingredients such as humectants, preservatives, buffers, and antioxidants which are useful as carriers in such formulations.

[0020] Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of formula I which is effective at such dosage or as a multiple of the same, for instance units containing 5 mg to 500 mg, usually around 10 mg to 250 mg.

[0021] The pharmaceutical compositions of the present invention may be prepared by the admixture of compound of formula (I) with a pharmaceutically acceptable carrier. Conventional pharmaceutical excipients may be admixed as required. Example of suitable formulations are given in the above-mentioned US Patent. No. 4,649,139.

[0022] The present invention can be used for treatment by the administration of a non-toxic effective amount of compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a composition as hereinbefore defined. The method is particularly suitable for the treatment of disorders in mammals that are susceptible to sodium channel blockers and antifolates, and particularly disorders such as epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for treatment of mammalian cancers; and for treatment of malaria.

[0023] The present invention also provides compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a composition as hereinbefore defined for, or for the preparation of, a medicament. The medicament is particularly suitable for treatment of disorders in mammals that are susceptible to sodium channel blockers and antifolates, and particularly disorders such as epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for treatment of mammalian cancers; and for treatment of malaria.

[0024] As indicated above, the compound of formula (I) is generally useful in treating such disorders by oral administration or intravenous injection.

[0025] The compound of formula (I) is normally administered at a dose of from 0.01 mg/kg to 20 mg/kg per day, preferably 0.1 to 5.0 mg/kg per day.

[0026] In view of the known use in humans of structurally similar compounds such as lamotrigine, and other known compounds similar to formula (I) no major toxicity problems are anticipated in use of the compound of formula (I). However appropriate testing procedures should be carried out before clinical use.

[0027] Aspects of the present invention will now be illustrated in further detail with reference to the accompanying examples. All compounds of the examples which are not covered by the claims are to be understood as reference examples and do not form part of the invention.

[0028] The methodology for preparation of the compound of formula (I) and other compounds used in testing, is reported below. This may be adapted to prepare analogous compounds with additional or alternative substituents or moieties.

[0029] In the procedures below all melting points are in ° C.

3,5-Diamino-6-Aryl-1,2,4-triazine compounds

[0030] 

3,4-Dimethoxybenzoyl cyanide (3; Ar = 3,4-dimethoxyphenyl)

[0031] A well stirred mixture [paddle stirrer] of 3,4 - dimethoxybenzoyl chloride [AcrosOrganics] (14.05g; 0.070mol), dry toluene (32cm³), dry acetonitrile (8.0cm³), copper I cyanide (8.5; 0.095mol) and Celite (5g) was heated under reflux until no acid chloride remained (~1.5hrs). The dark reaction mixture was cooled to ∼70° and diluted with toluene (150cm³). After stirring for an additional ∼30 minutes, the resulting slurry was filtered through a bed of chromatographic silica gel (~2.5cm) and the pale yellow filtrate evaporated in vacuo to constant weight to give the title compound as a lemon yellow solid. Yield = 11.41g (85.3%), Mpt = 143-145°C. The product was used directly in next stage.

Aminoguanidine bismesylate 4

[0032] To a stirred solution of 99.5% methanesulphonic acid [Aldrich] (422g; 4.40mol) in methanol (720cm³) at 40° was added portionwise over 30 minutes aminoguanidine bicarbonate [Aldrich] (272.0g; 2.00mol). When the addition was complete, the solution was stirred until the temperature had fallen to ~ 40° and then treated slowly with cold ether (500cm³). During the addition, colourless needles started to deposit. The resulting slurry was stood at 0° for 4hrs, filtered and the product washed with cold ether and dried overnight in vacuo at 50°. Yield = 528g (99.25%), mpt = 149 - 150° (Lit: WO/2004/026845; 147.5°)

Schiffs Base, cyanohydrazone (5, Ar = 3,4-dimethoxyphenyl)

[0033] To a stirred solution of aminoguanidine bismesylate (14.0g; 0.053mol) in 99.5% methanesulphonic acid (22g) at 65 - 70° was added dropwise a warm solution of 3,4 - dimethoxybenzoyl cyanide (5.7g; 0.030mol) in acetonitrile (30cm³) over -25 minutes. The mixture was then stirred at 68° until a sample gave a clear solution in water (~2.5hrs) and then poured onto crushed ice/water (125g) giving a pale yellow precipitate. The stirred mixture was neutralised (pH 8-9) with 48% sodium hydroxide (19.0cm³) giving a bright yellow precipitate. The product was filtered, washed with cold water and dried in vacuo at 45°. Yield = 6.21g (83.8%), Mpt = 98-100°C, TLC [SiO₂ plate, 10% methanol in chloroform], R_f = 0.52. The product was used directly in the next stage.

6-Alkyl/Aralkyl-3,5-diamino-1,2,4-triazine compounds

[0034] 

Triphenylacetyl chloride [3; R₁=R₂=R₃ = Ph]

[0035] A stirred mixture of triphenylacetic acid (21.7g; 0.075mol) and dry dimethylformamide (2 drops) in dry dichloromethane (100cm³) was treated with oxalyl chloride (14g; 0.11 mol) which was added in 4 approximately equal portions over -25 minutes. The mixture was stirred at 35° until evolution of hydrogen chloride had ceased (~4hrs). The resulting colourless solution was evaporated in vacuo at 40° to constant weight to give the title compound as a colourless crystalline solid. Yield = 23.24g (100.0%). The product was used directly in next stage.

[0036] Similarly prepared were:

Triphenylacetyl cyanide [4; R₁=R₂=R₃ = Ph]

[0037] A well stirred mixture [paddle stirrer] of triphenylacetyl cyanide (23.24g; 0.075mol), dry toluene (40cm³), dry acetonitrile (10cm³), copper I cyanide (9.20g; 0.103mol), Celite (3.5g) and finely powdered potassium iodide (2g) was heated under reflux until no acid chloride remained (~18hrs). The dark reaction mixture was cooled to ∼75° and diluted with toluene (150cm³). After stirring for an additional -30 minutes, the resulting slurry was filtered through a bed of chromatographic silica gel (~2.5cm) and the colourless filtrate evaporated in vacuo to constant weight to give the title compound as a colourless solid. Yield = 21.97g (98.7%), Mpt = 67-69°. The product was used directly in next stage.

Schiffs Base, cyanohydrazone, (4; R₁=R₂=R₃ = Ph]

[0038] To a stirred solution of aminoguanidine bismesylate (15.00g; 0.0564mol) in 99.5% methanesulphonic acid (22.5g) at 65 - 70° was added dropwise a solution of Triphenylacetyl cyanide (8.91g; 0.030mol) in acetonitrile (25cm³) over -25 minutes. The mixture was then stirred at 68° until a sample gave a clear solution in water (~28hrs) and then poured onto crushed ice/water (150g) giving a semi-solid colourless precipitate. The mixture was neutralised (pH 8-9) with 48% sodium hydroxide (17.5cm³) giving the title compound as cream granular solid. The product was filtered off, washed with water and dried in vacuo at 45°. Yield = 8.47g (80.0%), Mpt = 112-114°, TLC [SiO₂ plate, 10% methanol in chloroform], R_f = 0.68. The product was used directly in the next stage.

Triazine Compounds

[0039] 

5(3)-Amino-6-(2,3-dichlorophenyl)-2,3(2,5)-dihydro-3(5)-imino-2-(2,2,2-trichloroethyl)-1,2,4-triazine trifluoromethanesulphonate [CEN-216]: [compound of the invention]

[0040] 

[0041] 3,5-Diamino-6-(2,3-dichlorophenyl) -1,2,4-triazine (Lamotrigine) (0.9g; 3.50mmole), 2,2,2-trichloroethyl triflate (1.0g; 3.55mmole), butan-2-one (10cm³) and dimethlformamide (5 drops) were stirred at reflux for 25hrs under nitrogen.

[0042] The solution was evaporated to dryness and the tan residue crystallised from acetone to give a fawn coloured microcrystalline powder. Yield = 510 mg. Mpt - 236-238, tlc (10% methanol-chloroform), Rf = 0.38.

2,2,2-Trichloroethyl triflate

[0043] A mixture of 2,2,2-trichloroethanol (7.5g; 0.05mole) and triflic anhydride (14.1g; 0.05mole) was heated at 80°C for 60 minutes.

[0044] After cooling to room temperature, the reaction mixture was diluted with ether (100cm³) and extracted with ice cold 5% sodium hydrogen carbonate solution (3 x 50cm³), dried over anhydrous sodium sulphate, filtered and evaporated to dryness in vacuo below 20°C. A colourless oil resulted. This solidified to colourless prisms on standing at 4°C. Yield = 10.91g (77.8%) Melting point 28-30°C. The product is used directly without further purification.

2,2,-Dichloroethyl triflate

[0045] A mixture of 2,2-dichloroethanol (5.75g; 0.05mole) and triflic anhydride (14.1g; 0.05mole) was heated at 80°C for 60 minutes.

[0046] After cooling to room temperature, the reaction mixture was diluted with ether (100cm³) and extracted with ice cold 5% sodium hydrogen carbonate solution (3 x 50cm³), dried over anhydrous sodium sulphate, filtered and evaporated to dryness in vacuo below 20°C. A colourless oil resulted. The product is used directly without further purification.

2,2,2-Bromoroethyl triflate

[0047] A mixture of 2,2,2-trichloroethanol (14.15g; 0.05mole) and triflic anhydride (14.1g; 0.05mole) was heated at 80°C for 120 minutes.

[0048] After cooling to room temperature, the reaction mixture was diluted with ether (100cm³) and extracted with ice cold 5% sodium hydrogen carbonate solution (3 x 50cm³), dried over anhydrous sodium sulphate, filtered and evaporated to dryness in vacuo below 20°C. A colourless solid resulted. Melting point 41-43°C. The product is used directly without further purification.

Biological Testing

[0049] The compound of Formula (I), and various analogues, were tested for various activities as follows:

Screening strategy

[0050] The screening strategy is designed to select compounds with appropriate sodium channel blocking activity and low side effect liability. To this end all compounds are processed through the primary sodium channel assay (veratrine-evoked uptake of [¹⁴C]guanidine into rat forebrain synaptosomes) and IC₅₀ values computed from generated concentration-effect curves. In order to complement this data IC₅₀'s for selected compounds to inhibit binding of [³H]BTX-B are also measured.

[0051] Previous studies have shown that substituted triazines are potential inhibitors of DiHydroFolate Reductase (DHFR) activity (McCullough and Bertino 1971, Cashmore et al, 1975, Booth et al, 1987) and Sapse et al, 1994). Inhibitors of DHFR (such as Methotrexate) have been used for the treatment of various cancers (Suster et al, 1978 and Niculescu-Duvaz et al, 1982) as inhibition of this enzyme interferes with cell growth but because of this effect (on cell growth) inhibitors of DHFR may also be teratogenic (Skalko and Gold, 1974, Feldcamp and Carey, 1993 and Buckley et al, 1997). Should compounds be found which are potent inhibitors of DHFR then such compounds may, themselves, have potential as anticancer agents. Several methods are available for measurement of inhibition of DHFR activity and for this study we have examined effects of compounds to inhibit the binding of [³H] methotrexate (Myers et al, 1975 and Rothenberg et al, 1977).

[0052] Another common side-effect marker is inhibition of human Ether-a-go-go Related Gene potassium (hERG) potassium channel (Inward rectifying, I_Kr) activity which can be fatal due to heart failure brought about by development of long QT syndrome. A useful preliminary screen to assess potential to affect this channel is assessed by measurement of inhibition of the binding of [3H]astemizole to cell membranes expressing hERG. Selected compounds are tested for this activity by measurement of inhibition @ 10 µM. Assuming inhibition values lie between 10% and 90% it is possible to compute an extrapolated IC₅₀ for each compound.

[0053] The above screening cascade identifies compounds with appropriate sodium channel blocking activities that have a low(er) propensity for aforementioned side-effect liabilities. In order to develop these compounds further, some knowledge of their pharmacodynamic properties is required.

[0054] Sodium channel blockers, such as Sipatrigine, which both reduces the neurological deficit and infarct volume after middle cerebral artery occlusion in rats (Smith et al, 1997) and phenytoin, (which protect retinal ganglion cell death in an experimental model of glaucoma (Hains and Waxman, 2005) show neuroprotective efficacy in a range of models of nerve degeneration.. As failure of oxygen supply compromises both glycolysis and oxidative phosphorylation, ischaemic damage ultimately leads to electrical failure (nerve signalling) and pump failure (restoration of cellular membrane potentials). These failures (of electrical and ion pump activity) are associated with decreased local concentrations of ATP (Astrup et al 1981). Thus the effect of compounds to maintain concentrations of ATP in 0.4 mm slices of rat hippocampus following a severe metabolic insult was used.

Experimental procedures

Preparation of rat forebrain synaptosomes and homogenates

[0055] Experiments were performed using forebrain (whole brain less cerebellum/medulla) from Male Wistar rats weighing 175-250g. All efforts were made to reduce the number of animals used and all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and the European Community Council Directive of 24 November 1986 (86/609/EEC). Following killing of animals by stunning and decapitation, the forebrain (whole brain less cerebellum/medulla) was rapidly dissected and transferred to a weighed tube containing ice-cold 0.25M sucrose.

[0056] Synaptosomes (heavy and light mitochondrial fraction containing synaptosomes) were prepared by transferring the forebrain (of known wet weight) to a glass Potter vessel to which 9 volumes ice-cold 0.25M sucrose had been added and homogenising, using a teflon pestle, by 8 'up and down strokes' of a Braun Potter S motor driven homogeniser set to 900rpm. The resulting homogenate was centrifuged at 1036 x g at 4° for 10 min and the supernatant collected. The remaining pellet was resuspended, as above, in fresh ice-cold 0.25M sucrose and the centrifugation step repeated. The supernatant fractions were pooled and centrifuged at 40,000 x g (average) at 4° for 15 min and the resulting pellet resuspended in the appropriate assay buffer at a concentration of 20-25 mg wet weight per ml appropriate assay buffer.

[0057] Homogenates were prepared by transferring the known weight of forebrain to a cooled tube containing 9 volumes of ice-cold 50mM pH 7.4 HEPES buffer. The mixture was homogenised @ 4° by 3 x 5 sec bursts of an Ultra-Turrax^™ homogeniser set at maximum speed. The resulting homogenate was centrifuged at 40,000 x g (average) at 4° for 15 min and the supernatant discarded. The resulting pellet was resuspended in 9 volumes of fresh ice-cold pH 7.4 buffer (as above), the centrifugation step was repeated and the resulting pellet resuspended in the [³H]BTX-B binding buffer at a concentration of 20-25 mg wet weight per ml assay buffer.

[¹⁴C] guanidine flux and binding of [³H]BTX-B

[0058] Both assays were carried out using 14ml polypropylene test tubes to which a range of concentrations of the compounds under test were added. Test compounds were dissolved in DMSO and added to assays such that maximum concentration of DMSO did not exceed 2% v/v.

[¹⁴C]guanidine flux:

[0059] The [¹⁴C] guanidinine flux assay was measured using the method of Pauwels PJ et al (1986) but carried out @ 30° for 2½ min.

Reference:

[0060] 

Pauwels PJ, Leysen JE, Laduron PM. [3H]Batrachotoxinin A 20-alpha-benzoate binding to sodium channels in rat brain: characterization and pharmacological significance. Eur J Pharmacol. 1986 May 27;124(3):291-8.

Binding of [³H]BTX-B

[0061] [³H]BTX-B binding was carried out using the method described by Catterall et al (1981), except that both bovine serum albumin and TTX were omitted from the incubation medium.

Reference:

[0062] 

Catterall WA, Morrow CS, Daly JW, Brown GB. Binding of batrachotoxinin A 20-alpha-benzoate to a receptor site associated with sodium channels in synaptic nerve ending particles. J Bio. Chem. 1981 Sep. 10; 256(17): 8922-7.

Binding of [³H]Methotrexate

[0063] All steps were carried out at 4° (or on ice). Freshly dissected rat liver was dissected into 0.25M ice-cold Sucrose and subsequently homogenised (U-turrax) in 50 mM pH 6.0 phosphate buffer (10 ml/g tissue) containing 15 mM Dithiothreitol. The resulting homogenate was centrifuged @ 47,500 x g for 20 min and supernatant (filtered through cotton wool to remove fatty lumps) stored @ -80° before use (Rothenberg et al).

[0064] Inhibition of the binding of [³H]methotrexate to rat liver homogenate supernatant fractions were carried out essentially as described by Arons et al, 1975. Results were calculated, either as IC₅₀ values (see below) derived from concentration-effect curves or as percentage inhibition values determined by comparison with control and cold Methotrexate (10 µM final concentration) binding values.

Reference:

[0065] 

Elliot Arons, Sheldon P. Rothenberg, Maria da Costa, Craig Fischer and M. Perwaiz Iqbal; Cancer Research 35, August 1, 1975, 2033-2038,

Computation of IC₅₀ values

[0066] Data are presented as mean ± sem of number of experiments indicated in brackets. IC₅₀ values were obtained from radioligand displacement or guanidine flux inhibition curves by plotting log-₁₀ concentration vs bound ligand/guanidine uptake according the equation:-


where

y

= bound (dpm)

x

= log₁₀ compound concentration

Rmin

= lower asymptote (i.e. 100% inhibition)

Rsp

= upper asymptote - Rmin (i.e. specific binding)

n

= slope (log_e)

and

C =

IC₅₀ (i.e. concentration required to inhibit 50% of specific binding

Hippocampal slice assay

[0067] Neuroprotective efficacy was measured in 0.4 mm slices of rat hippocampus using the method described by Fowler and Li (1998)¹ except that lodoacetate (400 µM)² was used as the metabolic insult. Compounds (usually 30 µM) were always directly compared with tetrodotoxin (1 µM)³ for their ability to maintain slice concentrations of ATP following inhibition of glycolysis.

References:

[0068] 

1. Fowler J C, Li Y. Contributions of Na+ flux and the anoxic depolarization to adenosine 5'-triphosphate levels in hypoxic/hypoglycemic rat hippocampal slices. Neuroscience 1998, 83, 717-722.

2. Reiner PB, Laycock AG, Doll CJ. A pharmacological model of ischemia in the hippocampal slice. Neurosci Lett 1990; 119:175-8

3. Boening JA, Kass IS, Cottrell JE, Chambers G. The effect of blocking sodium influx on anoxic damage in the rat hippocampal slice. Neuroscience. 1989. vol 33 (2), 263-268.

Measurement of ATP and protein

[0069] Individual slices were disrupted by ultra-sonication and the resulting homogenates centrifuged @ 10000 x g for 5 min @ 4°. The supernatant was decanted into a fresh tube and any remaining supernatant removed by vacuum aspiration. The pellet was resuspended in 0.5 ml 0.1 M KOH by ultra-sonication and the resulting suspensions warmed with gentle agitation @ 37 ° for 30 minutes.

[0070] Concentrations of ATP were measured in 6 µl of supernatant by mixing with Luciferase reagent (ATPLite from Perkin Elmer) and measuring subsequent luminescence in a 96-well plate Counter.

[0071] Protein concentration was measured using BCA^™ protein assay (Pierce) withBovine Serum albumin as reference standard.

[0072] ATP concentrations were expressed as nmoles/ mg protein and neuroprotective indices (% protection) calculated by direct comparison with the effect of 1 µM TTX.

hERG:

[0073] Compounds were sent to MDS Pharma for measurement of their inhibition @ 10 µM concentration of the binding of [³H]astemizole to HEK-293 cells expressing human recombinant hERG. Making the assumption that binding slopes would be 1.0 IC₅₀ values could be calculated (see above) for compounds exhibiting between 5% and 95% inhibition of binding.

L-type calcium channels

[0074] Compounds were sent to MDS Pharma for measurement of their inhibition @ 10 µM concentration of the binding of [³H]nitrendipine to rat cerebral cortex membranes. Making the assumption that binding slopes would be 1.0 IC₅₀ values could be calculated (see above) for compounds exhibiting between 5% and 95% inhibition of binding.

Rat microsome stability

[0075] Compounds were sent to BioFocus for measurement of their stability @ 1 µM concentration following incubation with rat liver microsomes for 40 minutes @ 37°.

MES Methodology (Maximal ElectroShock)

[0076] Male Wistar rats provided by BioLasco Taiwan (under Charles River Laboratories Technology Licensee) were used. Space allocation for 5 animals was 45 x 23 x 21 cm. Animals were housed in animal cages and maintained in a controlled temperature (21 - 23oC) and humidity (50% - 70%) environment with 12hours light/dark cycles for at least three days in MDS Pharma Services - Taiwan Laboratory prior to use. Free access to standard lab chow for rats [MF-18 (Oriental Yeast Co., Ltd. Japan)] and reverse osmosis (RO) water were granted ad libitum. All aspects of this work including housing, experimentation and disposal of animals were performed in general accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D. C., 1996).

[0077] Test compounds were suspended/dissolved in 2% Tween 80 and dosed orally at a dose volume of 10ml/Kg to groups of 5 Wistar male rats weighing 180 +/- 20 g, one hour before maximal electroshock (MES, 60 Hz sine wave, 150 mA, 200 msec duration) was applied through corneal electrodes. Appearance of maximal electroshock-induced tonic convulsions (MES) was determined for each animal. 50 percent or more (50%) inhibition of tonic convulsions by test substance indicates significant anticonvulsant activity.

Equipment/chemicals:

[0078] Animal cage (Allentown, USA), Electronic shock generator (In-house, R. O. C.), Needle for oral administration (Natsume, Japan) and Rat scale (500 g, Yamato, Japan). Diphenylhydantoin sodium salt (Sigma, USA) and Tween 80 (Sigma, USA).

[0079] Test substances (CEN-216, CEN-145, CEN-148, CEN-152 and CEN-154) at 14, 42 or 100 mg/kg were administered orally to groups of 5 Wistar derived male rats weighing 180 ± 20 g,.

Results

[0080] Data from the various testing procedures is set out in the Table below:

	[14C]guanidine flux	[3H]mtx binding	hERG	hERG	L-type Ca2+	L-type Ca2+	Microsome stability (human)
CEN nr	Mean	IC50 (µM)	% inhibition @ 10 µM	IC50 (µM)	% inhibition @ 10 µM	IC50 (µM)	% metabolised
	IC50 (µM)	(% inhibition @ 125 µM)		(extrapolated from 10 µM inh'n)		(extrapolated from 10 µM inh'n)	(40 min incubation 37°)
		631(17 ± 2(4))
1 (Ltg)	219.2	11** 68*(***)	1	989	17	48.8	20
216			12	73.3	27	27.0	0
217			28	25.7
218			20	40.0

* 99µM ** 198µM *** uses fresh batch of supernatant Inhibition of binding of [3H]batrachotoxinin binding to rat (wistar) brain Data are presented as % inhibition @ 10 µM and extrapolated IC50's (which assumes hill slope = 1). Compounds which give < 5% inhibition are ascribed IC50's of > 200 µM Compounds which give > 95% inhibition are ascribed IC50's of < 0.5 µM

Inhibition of binding of [3H]BTX-B

Compound	% inhibition (@ 10 µm)	Extrapolated IC50 (µM)
CEN-1	-28	> 200
CEN-216	42	13.8
CEN-217	105	< 0.5
CEN-218	108	< 0.5
CEN-248	27	27

Summary of [3H]batrachotoxinin binding method - 279510 Sodium Channel, Site 2

Source:	Wistar Rat brain
Ligand:	5 nM [.H] Batrachotoxin
Vehicle:	1 % DMSO
Incubation Time/Temp:	60 minutes @ 37.C
Incubation Buffer:	50 mM HEPES, 50 mM Tris-HCl, pH7.4, 130 mM Choline
Chloride,	5.4mM KCI, 0.8 mM MgCl., 5.5 mM Glucose, 40 µg/ml LqTx
KD:	0.052 µM*
Non-Specific Ligand:	100 µM Veratridine
Bmax:	0.7 pmole/mg Protein *
Specific binding:	77%
Quantitation Method:	Radioligand Binding
Significance Criteria:	>/= 50% of max stimulation or Inhibition

Hippocampal slice data

Standard Compound	Conc'n (µM)	% protection (v 1 µM TTX) (mean ± sem)
TTX	1	100
Lamotrigine [CEN-001]	30	41 ± 5 (3)
DPH	30	48

MES Results for N-Alkyl substituted triazines

Compound	Dose (mg/kg po)	Results (% protection)
CEN-001 (Lamotrigine)	25	100
	10	100
	3	60
CEN-216	100	100
	30	100
	10	80
	3	0
	1	0
CEN-217	100	60
CEN-218	100	40
CEN-248	30	100
DiPhenylHydantoin	100	80

(n = 5 rats)

Compound No.	Structure	Nach ([3H]BTX-B) (extrapolated IC50 µM)	hERG ([3H]astemizole) (extrapolated IC50 µM)	Rat MES (anticonvulsant in vivo) (% protection @ mg/Kg (free base) po)	DHFR (% inhibition @ 125 µM)
CEN-216		20*	50*	80 @ 10 (ED50 = 9 mg/Kg po) (100 % protection @ 72 mg/Kg (free base) po @ 1 h, 6h and 24h prior to mouse MES). (i.e. long duration of action following po dose in mouse)	27
CEN-217		< 0.5	26	60 @ 100	ND
CEN-218		< 0.5	40	40 @ 100	ND
CEN-248		27	ND	100 % protection @ 30	87

CEN-216 extrapolated IC50 in L-type Calcium (phenylalkylamine site) binding assay = 27 µM (CEN-001) Lamotrigine result using same enzyme prep @ 125 µM gave 26% inhibition

[0081] In rat model of epilepsy (Maximal ElectroShock - MES), in which compounds were dosed orally one hour prior to 'shock', the ED50 (effective dose to protect 50% of rats from limb extension etc.) for CEN-216 is approx 9 mg/kg free base.

[0082] At 100 mg/kg free base in the above MES test CEN-217 and 218 gave 60% and 40% protection respectively i.e. ED50 for compounds of (very approx) 80 and 120 mg/kg free base respectively.

[0083] In mouse model of epilepsy (MES), compounds CEN-079 and CEN-216 were dosed orally (100 and 72 mg/kg free base respectively) @ 1h, 6h and 24h prior to 'shock). Both compounds showed significant protection at all time points in that 100 % protection was seen at all time points except that @ 24h CEN-079 protective effect had reduced to 40% protection

[0084] The screening data obtained in respect of representative compounds of the invention points to the suitability of compounds of general formula (I)) for treatment of disorders in mammals that are susceptible to sodium channel blockers and antifolates, and particularly disorders such epilepsy, multiple sclerosis, glaucoma and uevitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motorneurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for treatment of mammalian cancers; and for treatment of malaria.

Claims

1. The compound of formula I:

in which z is a single bond and wherein

A is 2,3-dichlorophenyl and R₁ is -CH₂CCl_3;

or a salt thereof.

2. A pharmaceutical composition comprising the compound as claimed in claim 1 or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable carrier.

3. A pharmaceutical composition as claimed in claim 2 for use in the treatment of epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; for use in treatment of mammalian cancers; or for use in treatment of malaria.

4. The compound as claimed in claim 1, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of: epilepsy, multiple sclerosis, glaucoma and uveitis, cerebral traumas and cerebral ischaemias, stroke, head injury, spinal cord injury, surgical trauma, neurodegenerative disorders, motor neurone disease, Alzheimer's disease, Parkinson's disease, chronic inflammatory pain, neuropathic pain, migraine, bipolar disorder, mood, anxiety and cognitive disorders, schizophrenia and trigeminal autonomic cephalalgias; of mammalian cancers; or of malaria.

5. The compound as claimed in claim 1, or a pharmaceutically acceptable salt or solvate thereof, or a composition as claimed in claim 2 or 3 for use in the treatment of epilepsy, multiple sclerosis, motor neurone disease or neuropathic pain.

Ansprüche

1. Verbindung der Formel I:

in welcher z eine Einfachbindung ist und worin

A 2,3-Dichlorphenyl ist und R¹ -CH₂CCl₃ ist;

oder ein Salz davon.

2. Pharmazeutische Zusammensetzung, umfassend die Verbindung wie in Anspruch 1 beansprucht oder ein pharmazeutisch verträgliches Salz oder Solvat davon, in Mischung mit einem pharmazeutisch verträglichen Träger.

3. Pharmazeutische Zusammensetzung wie in Anspruch 2 beansprucht zur Verwendung bei der Behandlung von Epilepsie, Multipler Sklerose, Glaukom und Uveitis, zerebralen Traumata und zerebralen Ischämien, Schlaganfall, einer Kopfverletzung, einer Rückenmarksverletzung, einem chirurgischen Trauma, neurodegenerativen Störungen, einer Motoneuronerkrankung, der Alzheimer-Krankheit, der Parkinson-Krankheit, chronischen entzündlichen Schmerzen, neuropathischen Schmerzen, Migräne, einer manisch-depressiven Psychose, affektiven Störungen, Angststörungen und kognitiven Störungen, Schizophrenie und trigemino-autonomen Kopfschmerzerkrankungen; zur Verwendung bei der Behandlung von Krebserkrankungen bei Säugern; oder zur Verwendung bei der Behandlung von Malaria.

4. Verbindung wie in Anspruch 1 beansprucht, oder ein pharmazeutisch verträgliches Salz oder Solvat davon, zur Verwendung bei der Behandlung von: Epilepsie, Multipler Sklerose, Glaukom und Uveitis, zerebralen Traumata und zerebralen Ischämien, Schlaganfall, einer Kopfverletzung, einer Rückenmarksverletzung, einem chirurgischen Trauma, neurodegenerativen Störungen, einer Motoneuronerkrankung, der Alzheimer-Krankheit, der Parkinson-Krankheit, chronischen entzündlichen Schmerzen, neuropathischen Schmerzen, Migräne, einer manisch-depressiven Psychose, affektiven Störungen, Angststörungen und kognitiven Störungen, Schizophrenie und trigemino-autonomen Kopfschmerzerkrankungen; von Krebserkrankungen bei Säugern; oder von Malaria.

5. Verbindung wie in Anspruch 1 beansprucht, oder ein pharmazeutisch verträgliches Salz oder Solvat davon, oder eine Zusammensetzung wie in Anspruch 2 oder 3 beansprucht zur Verwendung bei der Behandlung von Epilepsie, Multipler Sklerose, einer Motoneuronerkrankung oder neuropathischen Schmerzen.

Revendications

1. Composé de formule I :

dans lequel z est une simple liaison et dans lequel

A est 2, 3-dichlorophényle et R₁ est -CH₂CCl₃ ;

ou un sel de celui-ci.

2. Composition pharmaceutique comprenant le composé selon la revendication 1 ou un sel ou un solvate pharmaceutiquement acceptable de celui-ci, en mélange avec un support pharmaceutiquement acceptable.

3. Composition pharmaceutique selon la revendication 2, pour une utilisation dans le traitement de l'épilepsie, la sclérose en plaques, le glaucome et l'uvéite, les traumatismes cérébraux et les ischémies cérébrales, un accident vasculaire cérébral, une lésion de la tête, une lésion de la moelle épinière, un traumatisme chirurgical, des troubles neurodégénératifs, la maladie des motoneurones, la maladie d'Alzheimer, la maladie de Parkinson, une douleur inflammatoire chronique, une douleur neuropathique, la migraine, le trouble bipolaire, les troubles de l'humeur, de l'anxiété et cognitifs, la schizophrénie, les céphalées autonomes trigéminales ; pour une utilisation dans le traitement de cancers de mammifère ; ou pour une utilisation dans le traitement du paludisme.

4. Composé selon la revendication 1, ou un sel ou un solvate pharmaceutiquement acceptable de celui-ci, pour une utilisation dans le traitement de l'épilepsie, la sclérose en plaques, le glaucome et l'uvéite, les traumatismes cérébraux et les ischémies cérébrales, un accident vasculaire cérébral, une lésion de la tête, une lésion de la moelle épinière, un traumatisme chirurgical, des troubles neurodégénératifs, la maladie des motoneurones, la maladie d'Alzheimer, la maladie de Parkinson, une douleur inflammatoire chronique, une douleur neuropathique, la migraine, le trouble bipolaire, les troubles de l'humeur, de l'anxiété et cognitifs, la schizophrénie, et les céphalées autonomes trigéminales ; de cancers de mammifère ; ou du paludisme.

5. Composé selon la revendication 1, ou un sel ou un solvate pharmaceutiquement acceptable de celui-ci, ou composition selon la revendication 2 ou 3, pour une utilisation dans le traitement de l'épilepsie, la sclérose en plaques, la maladie des motoneurones ou une douleur neuropathique.